"We live in a world of color- color is in the trees and sky around us, in our clothes, and in our homes. When the colors of familiar things differ from what we expect, we are usually upset - greenish skies warrant of bad weather, black clothes denote mourning, intense colors in the home stimulate or agitate. The same applies to food. Consumers first judge the quality of the food product by its color." Food Technology, page 49 (July, 1986). However much one may decry a consumer attitude which places higher priority on visual impact than on gustatory and nutritional value, it is an attitude which must be actively confronted in the marketplace.
Although naturally occurring pigments perforce were the first used food colorants, the development of chemistry as a discipline led to many synthetic dyes, especially anilines, to supplant naturally occurring pigments as food additives. As a class synthetic colorants have many advantages, such as a uniform and reproducible color, color stability, absence of flavor, and an oxidative and/or thermal and/or light stability superior to naturally occurring pigments, broad availability relatively insensitive to changes in crop yields, and so forth. As a result, the popularity of synthetic colorants at least is understandable.
However, with heightened awareness of a consuming public to food additives and increased testing of some representative examples came a concern about their safety. Recent years have seen some materials formerly used as food colorants run the gamut from being beyond reproach to being suspect and even banned or at least used restrictedly. For example, FD&C Red No. 2 and FD&C Violet No. 1 have been banned in the United States and many other countries. Because of a variety of allergic reactions in sensitive individuals induced by FD&C Yellow No. 5 a recent ruling by the FDA requires food colored with it be declared as such on product labels. As a consequence the pendulum has begun to swing once more toward naturally occurring pigments as food additives.
The pigments produced by Monascus species fungi traditionally grown on rice in the Orient are orange and relatively insoluble in water, but readily react with compounds containing amino groups to form water soluble colorants. Monascus pigments have been used in the Orient for hundreds of years as a general food colorant and as a colorant for wine and bean curd. The pigments can be made water soluble or oil soluble, are stable at a pH range 2-10, are heat stable and can be autoclaved. In oriental countries microorganisms of this type typically are grown on grains of rice and once the grains have been penetrated by the red mycelium the whole mass is finely ground with the resulting powder used as a food colorant. The orange pigment is a mixture of monascorubrin and rubropunctatin, whose structures were elucidated by B. C. Fielding et al., Tetrahedron Letters No. 5, 24-7 (1960) and Kumasaki et al., Tetrahedron, 18, 1171 (1962), which differ in the former having a 7-carbon ketonic group and the latter having a 5-carbon ketonic group. For the purposes of this application, " Precursor pigment" refers to any mixture of water insoluble orange pigment containing monoascorubrin and rubropunctatin as produced by fermentation of a suitable Monascus species.
Commercial production of precursor pigment requires development of a suitable fermentation procedure, which has been the subject of many reports in recent years. Shepherd et al., U.S. Pat. No. 4,145,254, made an important advance by using a process with two phases in which the microorganism first was cultivated at pH 4-7 in a growth-promoting medium, then was transferred to a second medium at pH 2-4 to stimulate precursor pigment production. The low pH did not interfere with precursor pigment production but inhibited its subsequent reaction with amino groups of proteins and/or ammonium ions in the medium. The result was the exclusive production of orange precursor pigment as a colorant. As another example U.S. Pat. No. 4,442,209 claims to increase precursor pigment formation by cultivating a Monascus species in a medium containing maltitol.
As Shepherd et al. noted, ". . . if it is desired commercially to obtain a pigment having a perfectly determined structure which may be subjected to rigorous tolerance tests and which shows perfectly reproducible properties, it is the high-yield production of a high-purity orange pigment which should be researched in the first instance." All processes described to date retain serious disadvantages associated with the separation and isolation of high purity precursor pigment. More particularly, in the prior art processes precursor pigment is formed as an amorphous, clumpy mass, which is believed to be a consequence of some type of pigment-lipid association. This solid mass is not separable from the mycelium, as by partitioning into another phase; the strong association of the precursor pigment with fungal-produced lipids makes pigment extraction and purification very difficult, onrous, and costly. A perhaps typical prior art method utilizes the extraction of the pigment production medium with an organic solvent after separation of mycelium, or extraction of the production medium containing homogenized mycelium with an organic solvent. The solvent then is evaporated to afford crude precursor pigment which is subsequently further purified, as by chromatography and crystallization for example. Such an extraction-chromatography-crystallization procedure is inefficient as regards yield of the purified precursor pigment, costly because of the use of solvents to extract the pigment and the necessity of expanding energy to evaporate solvent from the extract, and not readily adaptable to continuous fermentation with continuous precursor pigment production.
Although there may be other aspects of precursor pigment production from Monascus which need attention, for example, obtaining suitable mutants or otherwise genetically altered microorganisms, this application is directed solely to an improvement in precursor pigment production which eliminates the need for costly extraction methods. Specifically, I have discovered that if during fermentation of pigment-producing Monascus species the medium contains certain added materials, precursor pigment is formed in a crystalline state, relatively pure per se and unassociated with fungal-produced lipids. Equally important is the observation that the precursor pigment crystals so formed can be readily partitioned into an oil phase, affording their facile separation from the mycelium and fermentation medium. The theoretical basis of the invention herein is uncertain, but it may be that certain materials act as biochemical regulators which block lipid synthesis while not affecting precursor pigment production. Whatever the theoretical basis, the effect of the addition of these biochemical modulators is that precursor pigment is excreted from the cell and rapidly forms large bright orange crystals which can be collected. The crystals are rather pure and ordinarily need not be further purified prior to their use. The process based on this discovery produces precursor pigments from glucose as a carbon source with 7% efficiency and is readily adapted to continuous fermentation. As will be readily recognized, the process also is independent of the particular microorganism producing the precursor pigment and the medium in which the microorganism is grown.